The present invention relates to a method for obtaining recombinant proteins, the origin of which is the membrane of the HIV virus responsible for the acquired immunodeficiency syndrome (AIDS), allowing the restoration of their native trimeric form, and to the use of these proteins for the purpose of vaccination or of diagnosis.
The HIV envelope glycoprotein is encoded by the xe2x80x9cenvxe2x80x9d gene, and the translation of the corresponding mRNA gives a glycosylated protein, gp160, in the form of a precursor with a molecular mass of 160 kDa. gp160 is cleaved inside the cell to give, at the cytoplasmic membrane during budding of the virus in the process of formation, on the one hand gp120, which is found on the outside of the cell and of the virus, and on the other hand gp41, which is the transmembrane portion of the glycoprotein and which corresponds to the carboxy-terminal end of the precursor. Once the viral particle has been released, gp41, the only transmembrane protein, will have its carboxy-terminal end turned toward the inside of the virus and its amino-terminal end projecting on the outside, maintaining itself associated noncovalently with gp120. It is attached noncovalently to gp41 via its amino-terminal end, while the rest of the protein is involved in the recognition of the CD4 receptor and of the CCR5 or CXCR4 coreceptors (specific for auxiliary T4 lymphocytes, macrophages; Trkola et al., J. Virol., 72, 1876-85, 1998; Schols et al., J. Virol., 72, 4032-4037, 1998; Rubbert et al., J. Immunology, 160, 3933-3941, 1998) The binding of gp120 to CD4 makes it possible to expose the membrane of the target cell to the amino-terminal hydrophobic portion of gp41, thereby inducing the mechanism of fusion of the viral and cell membranes, this fusion being the cause of the penetration of the virion into the target cell during infection (Wong-Staal et al., In Molecular Genetic Medecine, 2, Friedman ed., 189-219, 1992; Berger et al., Nature, 391: 240, 1998).
This process of recognition of the viral receptor, followed by the fusion of the membranes due to the interaction of the amino-terminal end of the fusion protein with the target cell membrane, is not a mechanism unique to HIV. It is made possible due to the presence, in oligomeric form, of the transmembrane glycoproteins of the virus. Bridging using chemical agents has made it possible to demonstrate trimers within the glycoproteins of the MuLV (Pinter et al., J. Virol., 30, 157-165, 1979), and MuMTV (Racevskis et al., J. Virol., 35, 937-948, 1980) envelope. It has also been shown that the RSV envelope protein forms oligomers which are found in infected cells and viral particles (Einfeld et al., Proc. Natl. Acad. Sci. USA, 85, 8688-8692, 1988). The influenza virus also expresses, at its surface, a haemagglutinin in trimeric form. In the latter case, the multimeric form is required for the intracellular transport of the protein (Copeland et al., J. Cell. Biol., 103, 1179-1191, 1986). The [lacuna] influenza also expresses, at its surface, a neuraminidase in the form of a tetramer (Varghese et al., Nature, 303, 35-40, 1983).
Although there is no doubt about the oligomeric nature of the various proteins encoded by the env gene, the monomer number has, itself, remained a controversial subject for a long time. The gp160 glycoprotein has, in fact, for a long time been described as being able to assemble into dimers or tetramers (Pinter et al., J. Virol., 63, 2674-2679, 1989; WO 94/00557 of the CNRS; Schawaller et al., Virology, 172, 367-369, 1989; Earl et al., Proc. Natl. Acad. Sci., 87, 648-652, 1990; Earl et al., J. Virology, 68, 3015-3026, 1994). Other more recent reports have, however, demonstrated that gp160 might, in fact, associate naturally, via its gp41 portion, in the form of trimers (Min Lu et al., Nature Structural Biology, 2, 1075-1082, 1995; Weisshorn et al., EMBO J., 15, 1507-1514, 1996; Weisshorn et al., Nature, 387, 426-430, 1997), the dimeric or tetrameric forms resulting, in fact, from aberrant interchain disulphide bridges or from transient oligomeric forms (see below).
For vaccinal purposes, the HIV envelope glycoprotein can be produced and purified, either by culturing the HIV virus on cell lines and purifying the glycoprotein from the culture medium (WO 94/00557 of the CNRS), or by expressing a recombinant of this protein using a vector other than HIV and purifying it from the culture medium (WO 91/13906, Chiron).
The purification of gp160 from cells infected with HIV makes it possible to obtain only tetramers, which is probably a transient oligomeric form, i.e. a form which does not correspond to that taken by its gp41 portion at the surface of the virus (WO 94/00557 of the CNRS).
The expression of a gp160 recombinant using a vector other than HIV, although having the advantage of escaping the dangers linked to the HIV infectious agent, does not make it possible also to have the xe2x80x9cnativexe2x80x9d oligomeric structure of gp160. Specifically, VanCott et al. have shown that the recombinant gp160 expressed by vaccinia, although having the power to adhere to CD4, comprises structural differences (J. Imm. Meth., 183, p. 114, col. 1, li. 19-22, 1995). Randall et al. have also shown that the recombinant gp160 expressed by vaccinia comprises aberrant interchain disulphide bridges (Virology, 179, 827-833, 1990).
Recently, Parren et al. have demonstrated a correlation between the production of antibodies which can neutralize, in vitro, HIV infection of cells and the oligomeric nature of gp120 (J. of Virology, 72, 3512-3519, 1998). For this, Parren et al. used a gp120 expressed by HIV in infected cells, probably in order to get round the problems linked to the structural differences between a native gp120, expressed at the surface of HIV, and those produced by expression vectors such as vaccinia.
Moreover, it is known that antibodies specific for the oligomeric structure of gp160 can be generated (Earl et al., above), and participate, in fact, in a neutralizing effect against HIV infection of cells, in vitro.
The present invention is directed towards providing a method for obtaining recombinant env gene expression products, which allows the restoration of their trimeric form, this form possibly being used in the context of a vaccination or in carrying out a diagnosis of HIV infection. In fact, the clinical trials carried out on recombinant gp160 molecules pose the problem of the spectrum of inhibition, which remains limited to only a few viral strains (Pialoux et al., Aids Res. Hum. Retr., 11, 373-381, 1995; Salmon-Cxc3xa9ron et al., Aids Res. Hum. Retr., 12, 1479-1486, 1995).
To date, although the trimeric form of a gp160 has been identified several times in a mixture of other polymeric forms, no-one has purified, nor suggested purifying, the trimeric form of gp160. The present [lacuna] is aimed at overcoming this need.
For this purpose, the invention relates to any purified recombinant glycoprotein which satisfies the following properties:
a) a capacity for adhesion to CD4;
b) an affinity with an anti-gp120 antibody capable of neutralizing HIV infection of cells, in vitro;
c) an affinity with an anti-gp41 antibody;
d) a trimeric form lacking interchain disulphide bridges.
A second subject of the present invention relates to a vaccine comprising the purified glycoprotein according to the invention, and an adjuvant.
A third subject of the present invention relates to the use of the glycoprotein according to the invention in the implementation of any method for diagnosing, in vitro, infections caused by HIV.
A final subject of the present invention relates to a method for obtaining a glycoprotein according to the invention, in which, by means of genetic recombination techniques, a glycoprotein satisfying the properties a), b) and c) according to the invention is expressed, purified and subjected to steps involving at least one reducing agent, one ionic detergent and/or one neutral detergent, under conditions such that a glycoprotein satisfying the conditions according to the invention is obtained.